One of the primary limitations in the use of non-viral gene transfer methods for gene therapy is the low efficiency of stable gene transfer in primary cells and tissues. Here we propose to incorporate an integrating mechanism to improve the efficiency of stable gene transfer by adapting for this purpose the Sleeping Beauty transposon (SB), newly generated from cloned genomic sequences of fish. Currently, SB has been demonstrated to mediate transposition in Hela cells and in fertilized embryos of zebrafish and Xenopus. To extend these studies, we will first characterize and quantitate SB-mediated transposition in cultured mammalian cells, including identification of rate-limiting components of transposition and assessment of transposition in non-dividing cells. These in vitro studies will establish the tools and conceptual basis for use of SB as a vector for gene therapy. As a direct assessment of the applicability of SB-mediated transposition to gene therapy, we will test this gene transfer system in the context of ex vivo and in vivo animal models for gene transfer. SB-mediated transposition into murine hematopoietic cells ex vivo will be tested, evaluating for reporter (green fluorescent protein) and drug-resistance (dihydrofolate reductase) gene transfer into committed progenitors and hematopoietic stem cells transplanted into recipient animals. In vivo SB-mediated transpostion into mouse liver will be tested, evaluating human alpha-1 antitrypsin reporter gene transfer and expression after systemic (i.e. tail vein) or portal vein injection. Results from these studies will establish proof of principle for use of the SB transposon as a vector for in vivo and ex vivo gene therapy strategies, setting the stage for potential future clinical application of SB to improve the stability of non-viral gene delivery.